Summary The rapid resealing of plasma membrane wounds is critical for cellular survival. Work supported by previous cycles of this grant showed that plasma membrane repair involves Ca2+-triggered exocytosis of lysosomes followed by massive endocytosis. Injury-induced release of lysosomal acid sphingomyelinase triggers a cholesterol/sphingolipid-dependent, clathrin-independent form of endocytosis, which involves caveolae or tubular invaginations that pinch off from the plasma membrane and carry lesions into cells for degradation. Clathrin-independent endocytosis has been described in many cell types, but there is considerable debate about how many independent pathways exist, and how they are initiated. By demonstrating that plasma membrane injury, Ca2+ influx and secretion of lysosomal acid sphingomyelinase trigger clathrin-independent endocytosis, our results have introduced much needed clarity to this field. The studies we now propose provide a unique opportunity for understanding how clathrin-independent endocytosis is regulated by plasma membrane injury, and how it promotes wound removal. Strikingly, our recent studies in B lymphocytes suggest that endocytosis-dependent plasma membrane repair and BCR-mediated B cell activation interfere with each other because of competition for lipid rafts, the cholesterol/sphingolipid-enriched plasma membrane microdomains that play a central role in clathrin-independent endocytosis and in BCR signaling and internalization after antigen capture. To understand the physiological impact of these findings, we will pursue two specific aims: 1) Characterize the injury-induced clathrin-independent form of endocytosis that promotes plasma membrane repair; 2) Examine the impact of plasma membrane wounding and repair on the regulation of B cell activation. These studies will significantly advance our understanding of clathrin-independent endocytosis and its role in plasma membrane resealing, and demonstrate how plasma membrane injury and repair regulate the function of B lymphocytes, cells that play an essential role in immune protection against infectious agents.